RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-811, 1998). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function.
The discovery of the Philadelphia Chromosome (Ph) represented the first consistent chromosomal abnormality causing a specific human cancer (Nowell P C et al., 1960, Science 132:1467). The Ph Chromosome is generated by a reciprocal translocation between the long arms of Chromosome 9 and Chromosome 22 (Rowley J D, 1973, Nature, 243:290-293.). It occurs in almost all patients with chronic myelogenous leukemia (CML), in about 10-20% of the adults with acute lymphoblastic leukemia (ALL) (Westbrook C A et al., 1992 Blood, 80:2983) and about 2% of patients with acute myelogenous leukemia (AML). The t(9;22) translocation fuses the Bcr gene from Chromosome 22 and the Abl gene from chromosome 9, resulting in the oncogenic Bcr-Abl fusion-gene (Heisterkamp N et al., 1983, Nature, 306:239). Variable breakpoints within the Bcr gene on chromosome 22 lead to the formation of different Bcr-Abl fusion gene variants which encode for different proteins p190Bcr-Abl (Mr 190,000), p210Bcr-Abl (Mr 210,000) and p230Bcr-Abl (Mr 230,000). In about 95% of the CML-patients the Bcr-Abl fusion transcripts e14a2 (former b3a2) and e13a2 (former b2a2) can be detected (reviewed in Barnes et al., 2002, Acta Haematologica, 108:180-202). The translated product is in each case a p210 kD Bcr-Abl protein. In patients with Ph+ALL a shorter transcript version called Bcr-Abl-e1a2, predominates (reviewed in Faderl et al., 2003, Cancer, 98:1337). Translation of this variant results in the somewhat lighter p190Bcr-Abl protein. Both Bcr-Abl proteins p190Bcr-Abl and p210Bcr-Abl are characterised by a dramatically increased tyrosine-kinase activity, as compared to that of normal Abl protein, leading to aberrant phosphorylation of downstream target molecules.
The kinase activity of Bcr-Abl can be inhibited by a specific tyrosine kinase inhibitor, Imatinib mesylate (STI571, Glivec), which is effective for treatment of Ph+leukemia (reviewed in Kurzrock et al., 2003, Ann. Intern. Med., 138:819). Nevertheless, both ALL and advanced CML patients frequently develop drug resistance after initial response predominantly caused by genetic abnormalities such as point mutations in the Bcr-Abl kinase domain or overexpression of Bcr-Abl (for review: Rothberg, 2003, Leukemia Res., 27:977). Therefore the development of alternative strategies to inhibit Bcr-Abl becomes increasingly important.
The breakpoint of the Bcr-Abl mRNA represents a unique and leukemia-specific nucleotide sequence. Such fusion transcripts encoding oncogenic proteins represent ideal targets for a disease-specific RNAi approach. The possibility to use RNAi for the specific degradation of the Bcr-Abl-e14a2 transcript variant, as well as other oncogenic fusion proteins, has been demonstrated recently (Wilda et al., Oncogene 2002, 21:5716; Scherr et al., Blood 2003, 101:1566; Heidenreich et al., Blood. 2003, 101:3157, Wohlbold et al., Blood 2003, 102:2236; Ritter U, et al., Oligonucleotides 2003, 13:365; Li et al., Oligonucleotides 2003, 13:401; Chen J, et al., J Clin Invest. 2004, 113:1784). The results presented are inconclusive, as Wilda et al. did not observe a sensitizing effect towards imatinib mesylate on Bcr-Abl-expressing cells by treatment with Bcr-Abl-specific siRNAs, whereas others did observe such effects. It was therefore unclear so far, whether the expression of two relevant Bcr-Abl transcripts other than the e14a2 transcript variant (e13a2 and e1a2) can be downregulated by an RNAi approach.
The present invention advances the art by providing methods and medicaments encompassing short dsRNAs leading to the down-regulation of p210Bcr-Abl and p190Bcr-Abl protein levels in murine 32D cells expressing the respective Bcr-Abl gene variants, in human leukemic MEG-01, K562 and SUP-B15 cells, and in cells freshly isolated from human subjects suffering from leukemia. These methods and medicaments may be used in research into, and in the treatment of, certain cancers.